Review article
Thermoresponsive hydrogels in biomedical applications
Leda Klouda,Antonios G.Mikos
*
Department of Bioengineering,Rice University,Houston,TX,USA Received 17November 2006;accepted in revised form 16February 2007
Available online 18July 2007
Abstract
Environmentally responsive hydrogels have the ability to turn from solution to gel when a speci?c stimulus is applied.Thermorespon-sive hydrogels utilize temperature change as the trigger that determines their gelling behavior without any additional external factor.These hydrogels have been interesting for biomedical uses as they can swell in situ under physiological conditions and provide the advan-tage of convenient administration.The scope of this paper is to review the aqueous polymer solutions that exhibit transition to gel upon temperature change.Typically,aqueous solutions of hydrogels used in biomedical applications are liquid at ambient temperature and gel at physiological temperature.The review focuses mainly on hydrogels based on natural polymers,N -isopropylacrylamide polymers,poly(ethylene oxide)–b -poly(propylene oxide)–b -poly(ethylene oxide)polymers as well as poly(ethylene glycol)-biodegradable polyester copolymers.
ó2007Elsevier B.V.All rights reserved.
Keywords:Thermosensitive hydrogels;In situ gelation;Drug delivery;Tissue engineering;Lower critical solution temperature
1.Introduction
Hydrogels are composed of hydrophilic homopolymer or copolymer networks and can swell in the presence of water or physiological ?uids.Chemical crosslinks (covalent bonds)or physical junctions (e.g.secondary forces,crystal-lite formation,chain entanglements)provide the hydrogels’unique swelling behavior and three-dimensional structure [1–3].Hydrogels have been a topic of extensive research in the past decades and their properties as for example their high water content and the possible control over the swell-ing kinetics make them very attractive for biomedical applications.More speci?cally,in situ forming hydrogels can provide a means for simple,‘‘custom-made’’therapeu-tics and diagnostics.A polymer solution can be prepared and allowed to gel in situ ,after photopolymerization [4,5],chemical crosslinking [6,7],ionic crosslinking [8]or in response to an environmental stimulus such as tempera-ture,pH or ionic strength of the surrounding medium [9,10].Hydrogels that respond to temperature change are the subject of this review.Their sensitivity to the thermal environment is useful as temperature is the sole stimulus for their gelation with no other requirement for chemical or environmental treatment,and can be thus produced e.g.upon injection to the body,when temperature is increased from ambient to physiological.
The phenomenon of transition from a solution to a gel is commonly referred to as sol–gel transition.Some hydrogels exhibit a separation from solution and solidi?cation above a certain temperature.This threshold is de?ned as the lower critical solution temperature (LCST).Below the LCST,the polymers are soluble.Above the LCST,they become increasingly hydrophobic and insoluble,leading to gel for-mation.In contrast,hydrogels that are formed upon cooling of a polymer solution have an upper critical solution temper-ature (UCST)[10].The sol–gel transition of thermosensitive hydrogels can be experimentally veri?ed by a number of techniques such as spectroscopy [11–13],di?erential scan-ning calorimetry (DSC)[11,12]and rheology [11].
There are various mechanisms behind thermogelation in aqueous solutions,and for some polymers they are still a
0939-6411/$-see front matter ó2007Elsevier B.V.All rights reserved.doi:10.1016/j.ejpb.2007.02.025
*
Corresponding author.Department of Bioengineering,Rice Univer-sity,P.O.Box 1892,MS-142,Houston,TX 77251-1892,USA.Tel.:+17133485355;fax:+17133484244.
E-mail address:mikos@https://www.doczj.com/doc/be17695989.html, (A.G.Mikos)https://www.doczj.com/doc/be17695989.html,/locate/ejpb
Available online at https://www.doczj.com/doc/be17695989.html,
European Journal of Pharmaceutics and Biopharmaceutics 68(2008)
34–45
topic of debate.Many polymers show a decrease in solubil-ity that is attributed by changes in the overall hydrophilic-ity of the polymer chains upon temperature change.When a polymer is dissolved in water,there are three types of interactions that take place:between polymer molecules, polymer and water and between water molecules.For poly-mers exhibiting an LCST,temperature increase results in a negative free energy of the system which makes water–polymer association unfavorable,facilitating the other two types of interactions.This negative free energy(D G) is attributed to the higher entropy term(D S)with respect to the increase in the enthalpy term(D H)in the thermody-namic relation D G=D HàT D S.The entropy increases due to water–water associations which are the governing interactions in the system.This phenomenon is the so-called hydrophobic e?ect[11,14,15].Polymer micelle packing[16]and coil to helix transition causing network formation[17]are examples of the conformational changes that take place at the critical solution temperature.All of these result in a reversible physical linking of the polymer chains,and gels can therefore return to solution after the thermal stimulus that caused their gelation is removed.
This article reviews the applications of thermosensitive hydrogels in?elds of interest for pharmaceutical and biomedical scientists and engineers.It emphasizes mainly the use of hydrogels based on natural polymers,N-isopro-pylacrylamide(NiPAAM)polymers,poly(ethylene oxide)–b-poly(propylene oxide)–b-poly(ethylene oxide)(PEO–PPO–PEO)as well as poly(ethylene glycol)(PEG)-biodegradable polyester copolymers.As there are some excellent reviews [18,19]summarizing the literature on thermosensitive hydrogels published in the past years,the scope of this work is to cover the advances in the?eld from2004until today(2006)(Fig.1).
2.Natural polymers and derivatives
Many natural polymers have been shown to exhibit gelation upon temperature change.Researchers have used them alone or in combination with synthetic polymers to fabricate thermally responsive hydrogels with desired properties.
2.1.Polysaccharides
2.1.1.Cellulose derivatives
Cellulose is a natural polysaccharide which is insoluble in water.Substitution of the hydroxyl groups on cellulose with more hydrophobic units as methyl or hydroxypropyl groups renders the originally insoluble cellulose water sol-uble[11].Methylcellulose(MC)is a cellulose derivative that has been extensively investigated for biomedical applications.It has thermoreversible gelation properties in aqueous solutions,gelling at temperatures in the range of60–80°C and turning into a solution upon cooling [11,20].Liu et al.[21]have grafted methylcellulose with the synthetic N-isopropylacrylamide(NiPAAm),combin-ing the thermogelling properties of both materials.It was possible to prepare fast reversibly thermogelling hydrogels by adjusting the ratios of the two components.They reported that a low percentage of methylcellulose decreases the LCST as compared to pNiPAAm,but with a high MC ratio the LCST increases.They also found that addition of MC to NiPAAM polymers enhances the mechanical strength of the hydrogel with no syneresis.
Another interesting approach towards a thermosensitive hydrogel system based on methylcellulose was recently reported by Stabenfeldt et al.[22].As it had been shown before[23]that methylcellulose showed low protein adsorption and cell adhesion,Stabenfeldt et al.functional-ized methylcellulose with the protein laminin,aiming towards the creation of a bioactive sca?old for neural tissue engineering.In order to facilitate laminin tethering,meth-ylcellulose was oxidized previous to functionalization. They reported that the laminin-functionalized oxidized methylcellulose(OXMC+LN)hydrogel promoted neuro-nal cell adhesion and showed higher cell viability rates than methylcellulose(MC),oxidized methylcellulose(OXMC) or laminin-functionalized methylcellulose(MC+LN) (Fig.2).Moreover,the concentration could be adjusted so that the hydrogel exhibits a lower critical solution tem-perature slightly below physiological temperature.
2.1.2.Chitosan
Chitosan is produced with the deacetylation of chitin, which can be found in the outer skeleton of shrimp and insects,among others.We would like to refer the reader to the excellent review by Garie′py and Leroux[19]which covers the developments of thermosensitive chitosan-pol-yol salt hydrogels until2004.More recently,Bhattarai et al.[24]incorporated poly(ethylene glycol)(PEG)into chitosan and were able to form a thermoreversible hydro-gel with no additional crosslinking agents.Moreover, PEG grafting improved the solubility of chitosan in water, and the gelation was found to be possible in physiological pH values.The same group[25]evaluated the PEG-grafted chitosan for controlled drug release in https://www.doczj.com/doc/be17695989.html,ing albumin as a model protein,an initial burst release was observed, which was followed by a steady release from the hydrogel for about3days.After this time,the remaining albumin could not be released from the hydrogel until the gel matrix was dissolved in the media.When the PEG-grafted chito-san was crosslinked in situ with genipin,a crosslinking agent with low cytotoxicity,quasi-linear drug release was possible for up to40days;however the hydrogel lost its thermoreversibility at37°C.
Chitosan-based hydrogels have been also investigated as potential cell carriers for tissue engineering applications.A copolymer of NiPAAm and water-soluble chitosan was tested for chondrogenic di?erentiation of human mesen-chymal stem cells(hMSC).The hydrogel showed a stable gelation at37°C and di?erentiation of hMSC into chon-drocytes was observed,both in vitro and in vivo.This hydrogel could be used for a minimally invasive treatment
L.Klouda,A.G.Mikos/European Journal of Pharmaceutics and Biopharmaceutics68(2008)34–4535
of vesicouretral re?ux with an endoscopic procedure through a single injection[26].Dang et al.[27]modi?ed chitosan by adding hydroxybutyl groups to its hydroxyl and amino reactive sites,rendering it water soluble and thermally responsive.The resulting hydrogel could gel within seconds after exposure to physiological temperature and was su?ciently strong to be handled with,as for exam-ple to measure mechanical properties.Upon cooling,the gel returned to liquid state.Having as an ultimate goal the treatment of degenerative disc disease,the hydrogel’s interaction with hMSC and cells derived from the interver-tebral disc was tested.The cells were able to proliferate and produce extracellular matrix when encapsulated in the hydrogels for a period up to2weeks.
A chitosan-glycerophosphate salt(GP)hydrogel was recently tested for its potential in neural tissue engineering.
36L.Klouda,A.G.Mikos/European Journal of Pharmaceutics and Biopharmaceutics68(2008)34–45
This thermally responsive hydrogel,as developed initially by Chenite et al.[28],has been shown to have good bio-compatibility in vitro ,but it had not been yet tested with nerve cells.Crompton et al.[29]compared the hydrogel to polylysine-functionalized chitosan-GP,with the hypoth-esis that the peptide polylysine might improve neuronal adhesion and neurite outgrowth.When neurons were grown in a two-dimensional culture,the functionalized hydrogel did not seem to signi?cantly a?ect cell survival as compared to chitosan-GP,but it was observed that increasing concentrations of polylysine inhibited neurite outgrowth.However,when the cells were cultured in a three-dimensional functionalized gel,a geometry that is more representative of the extracellular matrix (ECM)
environment,in certain polylysine concentrations more cells were viable than on non-modi?ed chitosan-GP.The authors concluded that polylysine-chitosan-GP may be a good candidate for neural tissue engineering.
2.1.
3.Dextran
A modi?ed precursor of the enzymatically biodegrad-able dextran (Dex)was formed by reaction with maleic anhydride (MA)and the Dex-MA polysaccharide was given thermoresponsive properties by photocrosslinking it with NiPAAm.The resulting hydrogel was partially biode-gradable and exhibited a higher LCST than pNiPAAm due to the hydrophilic and biodegradable nature of Dex-MA.Additionally,the carboxylic end groups of Dex-MA
render
Fig.2.Viability of neurons plated within methylcellulose sca?olds.(A)Representative micrograph images from 100-l m thick confocal scans of 3-D neuronal cultures in MC,OXMC,MC +LN,and OXMC àLN at 1day post-plating.Live cells:green,Dead cells:red.Scale bar =50l m.(B)A signi?cant increase in cell viability as indicated by #was observed with OXMC àLN when compared to MC,OXMC,and MC +LN,which did not show statistically signi?cant di?erences from each other (p <0.01).Reproduced from [22]with permission.(For interpretation of the references in color in this ?gure legend,the reader is referred to the web version of this article.)
L.Klouda,A.G.Mikos /European Journal of Pharmaceutics and Biopharmaceutics 68(2008)34–4537
the hydrogel pH sensitive[30].Another approach based on a dextran polysaccharide was reported by Huang et al.[31].
A dextran macromer containing oligolactate and2-hydroxyethyl methacrylate units(Dex-lactate-HEMA), which has hydrolytically degradable blocks,was copoly-merized with NiPAAm.This hydrogel showed an LCST close to that of pNiPAAm(approximately32°C).Its swell-ing and degradation in phosphate bu?ered saline(PBS) were studied at25and37°C.At25°C,which is below the LCST,the hydrogels had disintegrated within2weeks, with the rate of dissolution depending on their composi-tion.At37°C however,the degradation was much slower due to increased hydrophobic e?ects.Interestingly,when the hydrogel was tested for drug delivery,it was shown that a low molecular weight drug(methylene blue)was released slower at25°C than at37°C,whereas the opposite was observed for a high molecular weight substance(bovine serum albumin–BSA)(Fig.3).The authors concluded that the drug release pro?le depends on a number of factors,as the temperature,the swelling and degradation characteris-tics of the hydrogel,as well as the interactions of the drug and the hydrogel macromolecules.
2.1.4.Xyloglucan
Xyloglucan is a cytocompatible polysaccharide and has exhibited thermally responsive behavior when more than 35%of its galactose residues are removed[32].Xyloglucan gels have been used as a drug delivery vehicle for various applications[19],however there are not many data on the rheological and morphological characteristics of these hydrogels.Nisbet et al.[33]have examined the gelation properties of xyloglucan hydrogels as well as their mor-phology under physiological conditions.The gelation pro-cess seemed to be in?uenced by the presence of ions in PBS as compared to deionized water.As to the optimum con-centration,it was found that3%(wt)xyloglucan in aque-ous media possesses an elastic modulus that is signi?cantly higher than other natural or synthetic hydro-gels.Moreover,this concentration yielded a gel that could be freeze-dried and examined with scanning electron microscopy.The images showed a macroporous,intercon-nected,three-dimensional network.
2.2.Proteins
2.2.1.Gelatin
Gelatin is another biopolymer with thermoreversible properties.At temperatures below25°C,an aqueous gela-tin solution solidi?es due to the formation of triple helices and a rigid three-dimensional network.When the tempera-ture is raised above approximately30°C,the conformation changes from a helix to the more?exible coil,rendering the gel liquid again[17].As the opposite thermal behavior is desired for biomedical applications,researchers have com-bined gelatin with other polymers,which show thermal gelation close to body temperature.Gelatin has the advan-tage of allowing for easy modi?cation on the amino acid level;moreover,it is biodegradable and biocompatible [34].A binary-component hydrogel composed of gelatin and monomethoxy poly(ethylene glycol)–poly(D,L-lactide) (mPEG–DLLA)block copolymers was synthesized by Yang and Kao[34].For most compositions of gelatin and mPEG-DLLA,the hydrogel was shown to?ow at37°C and gel at room temperature,however a100mg/mL gelatin solution underwent fast gelation at37°C when mixed with 30%wt mPEG–DLLA.Di?erent hydrogel compositions were also examined for drug release kinetics with gentamy-cin sulfate as the model drug.At room temperature,5days or longer was necessary for50%drug release,and the release lasted up to40days.At37°C,gelatin-mPEG-DLLA showed an even slower release pro?le,however after1week the release was no longer detectable due to degradation of the hydrogel matrices.
Ohya and Matsuda[35]have grafted gelatin with NiP-AAm in an e?ort to produce a thermoresponsive extracel-lular matrix analogue.Aqueous solutions showed a sol–gel transition at physiological temperature when the weight ratio of pNiPAAm to gelatin chains was higher than5.8. Smooth muscle cells were suspended in medium solutions of pNiPAAm/gelatin and subsequently incubated
at Fig.3.Fractional release from a hydrogel with5:4NiPAAm:Dex-lactate-HEMA weight ratios in PBS of methylene blue(a)and BSA(b)at25(s)and37 (d)°C.Reproduced from[31]with permission.
38L.Klouda,A.G.Mikos/European Journal of Pharmaceutics and Biopharmaceutics68(2008)34–45
37°C.It was shown that a low hydrogel concentration (5%w/v)and a high pNiPAAm to gelatin ratio (P/G)sup-ported the highest cell proliferation and extracellular matrix production.The authors suggested that this was due to increased hydrophobicity caused by higher pNi-PAAm ratios,which would lead to the formation of large aggregates.As a result,a higher porosity with larger pore size occurs,which comprises a favorable cell environment (Fig.4).
Another protein-based hydrogel was proposed by Gil et al.[36].Gelatin was blended with silk ?broin to yield a thermoresponsive gel,which was stabilized at 37°C by the presence of b crystals of silk ?broin.The swelling pro-?le at temperatures below and above the helix-to-coil trans-formation of gelatin was evaluated,as well as the protein release from the matrices.The gel showed a higher swelling at physiological temperatures as compared to 20°C,but also higher mass loss due to dissolution and release of gelatin.
3.N -Isopropylacrylamide-based systems
Hydrogels based on poly(N -isopropylacrylamide)(pNiPAAm)and its copolymers belong to the most inten-sively investigated thermoreversible systems.Recent developments on pNiPAAm-based hydrogels include their use for drug delivery [12,13,37,38],cell encapsulation and delivery [39,40]and cell culture surfaces [41].Poly(N -iso-propylacrylamide)is non-biodegradable and exhibits a sharp phase transition,with an LCST at about 32°C in pure water [37,42].Below the LCST,pNiPAAm assumes a ?exible,extended coil conformation in aqueous solu-tions.At the LCST,it becomes hydrophobic and the poly-mer chains seem to collapse prior to aggregation in globular structures [14,43].Copolymerization of NiP-AAm with a more hydrophilic monomer increases the overall hydrophilicity of the polymer,and the stronger polymer–water interactions lead to an increase in the LCST.Likewise,copolymerization with a more hydro-phobic monomer results in a lower LCST than pNiPAAm [44].Moreover,the phase transition temperature is in?u-enced by the presence of salts [45]and pH to a certain extent [42,45].
Coughlan and colleagues [38]evaluated the swelling and release pro?le of crosslinked pNiPAAm hydrogels as a function of the physicochemical properties of the loaded drugs.Dried hydrogel discs were loaded by sorption of a drug solution,the solvent was removed and the hydrogels were allowed to swell in a bu?er solution.Hydrogel swell-ing was decreased in the presence of hydrophobic drugs and the opposite e?ect was observed for hydrophilic drugs.In temperatures above the LCST,the system showed con-traction and deswelling,and a solubility-dependent drug pulse release was shown for hydrophobic drugs,whereas hydrophilic drugs showed a molecular weight-dependent drug pulse.The authors suggested that drug properties such as solubility,size and chemical nature should be con-sidered when a thermosensitive hydrogel as pNiPAAm is chosen as a delivery vehicle.
Copolymers of NiPAAM have also been popular in an attempt to yield hydrogels with thermal responsiveness and improved properties.The hydrogel potential of NiP-AAM copolymers with acrylic (AA)[12,39]and propylac-rylic acid (PAA)[13]was examined.The thermoreversible p(NiPAAm-co -AA)hydrogel was tested as a cell and drug delivery vehicle.Chondrocytes,dexamethasone and ascor-bate as di?erentiation factors as well as transforming growth factor b 3(TGF-b 3)were encapsulated in the hydrogel and were implanted subcutaneously in mice.The chondrogenic factors were provided in order to hinder chondrocyte de-di?erentiation in vivo .After 8weeks,signif-icant collagen II expression as well as proteoglycan
and
Fig.4.Structure of pNIPAAm-gelatin (P/G)hydrogels as a function of P/G ratio and concentration.Reproduced from [35]with permission.
L.Klouda,A.G.Mikos /European Journal of Pharmaceutics and Biopharmaceutics 68(2008)34–4539
polysaccharide production was evident,indicating that the cells had preserved their phenotype.This hydrogel in com-bination with the di?erentiation and growth factors holds promise for cartilage tissue engineering[39].Liu et al.
[12]have synthesized p(NiPAAm-co-AA)and polymerized it with ethyl acrylate(EA)using the interpenetrating poly-mer network(IPN)technology.An interpenetrating poly-mer network is formed by hydrophilic and hydrophobic networks that are only physically interconnected,without any chemical bonding,so that individual components retain their original properties.The same group[46]had found that IPN structures are e?ective amphiphilic drug carriers.A pH dependence on the swelling ratio of p(NiPAAm-co-AA)as well as of interpenetrating polymer network with ethyl acrylate(p(NiPAAm-co-AA)/pEA IPN)was observed at37°C.This was attributed to the presence of the carboxyl group on the acrylic acid.Swelling was lower on the IPNs due to the hydrophobic pEA.The drug release kinetics of both hydrogels were evaluated using daidzein as a model drug.P(NiPAAm-co-AA) showed an initial burst release,which was not observed on p(pNiPAAm-co-AA)/pEA IPN.It was concluded that the pEA chains in the IPN structure had a favorable e?ect in maintaining a slower and more stable release pro?le[12]. Also,copolymers of NiPAAm with propylacrylic acid (PAA)show a temperature and pH-sensitive behavior. Yin et al.[13]synthesized copolymers by a reversible addition fragmentation transfer(RAFT)method,using di?erent NiPAAm and PAA ratios.They showed that even small changes in pH can have a big e?ect on the LCST of the hydrogel.This feature can be useful for applications such as drug delivery,where physiological temperature and local pH di?erences can both act as stimuli,and for molecular switching over a desired pH range.
An interesting approach to a combination of stimuli-responsive attributes was recently proposed by Xu et al.
[47]in the form of a triblock copolymer hydrogel.A poly ((2-dimethyl amino)ethyl methacrylate-co-2-hydroxyethyl methacrylate)–b-poly(N-isopropylacrylamide)–b-poly((2-dimethyl amino)ethyl methacrylate-co-2-hydroxyethyl methacrylate)or p(DMAEMA-co-HEMA)-b-p(NiPAAm)-b-p(DMAEMA-co-HEMA)copolymer was synthesized by atom transfer radical polymerizations(ATPR).The hydroxyl groups on HEMA allowed for chemical cross-linking with glutaraldehyde.The hydrogel showed combined characteristics of its building blocks:its temper-ature-responsive behavior was attributed to pNiPAAm and the pH-sensitivity to pDMAEMA.
Our group[40]has synthesized thermogelling macro-mers for fabrication of a hydrogel for orthopedic tissue engineering applications.The aim was to yield a gel with better mechanical properties than most hydrophilic inject-able hydrogels.This was accomplished by incorporating a hydrophobic domain that provides cohesive interactions as well as functional groups for chemical crosslinking.A random copolymer of pentaerythritol monostearate diacrylate(PEDAS),N-isopropylacrylamide(NiPAAm),acrylamide(AAm)and2-hydroxyethyl acrylate(HEA) was synthesized.PEDAS contains a lipophilic side chain, and AAm and HEA can modulate hydrophilicity and add groups for subsequent acrylation and crosslinking.The thermal gelation is attributed to the NiPAAm block. Our studies so far have shown that the macromers possess a thermoreversible behavior.Future work is directed into further development and characterization of the hydrogel and examination of its potential in bone regeneration.
Another issue that has to be addressed is biodegradabil-ity.Many homo-and copolymers of NiPAAm are not bio-degradable[48],a fact that may prove problematic for some biomedical engineering applications.Nakayama and colleagues[37]have prepared thermally responsive, biodegradable polymeric micelles for controlled drug release.A hydrophobic block in the micelles was used to incorporate water-insoluble drugs.By combining a poly(N-isopropylacrylamide-co-N,N-dimethylacrylamide) (p(NiPAAm-co-DMAAm))block,which has an LCST around40°C,with poly(D,L-lactide),poly(e-caprolactone) or poly(D,L-lactide-co-e-caprolactone),which are all biode-gradable and hydrophobic,the group was able to fabricate polymeric micelles with controlled dimensions and phase transition temperatures.Below the LCST,the thermore-sponsive block forms the outer shell of the micelle,but upon temperature increase above the LCST,the block becomes increasingly hydrophobic and shrinks.In the case of p(NiPAAm-co-DMAAm)-b-p(D,L-lactide-co-e-caprolac-tone)diblock copolymer,temperature increase above the LCST proved to facilitate drug release(Fig.5).
Polymers based on pNiPAAm have found applications in another?eld crucial to biomedical scientists:their ther-moresponsive behavior has been proven useful in cell cul-ture substrates.By introducing a pNiPAAm layer on tissue culture plates,the hydrophilicity of the substrate can be modulated with temperature switch.It is well known that most cells preferentially adhere to hydropho-bic surfaces.Above its LCST(32°C),pNiPAAm shows a hydrophobic behavior.It represents therefore a suitable surface for cell attachment and proliferation at physiolog-ical temperature.By lowering the temperature below the LCST,the culture surface becomes hydrophilic and the cells automatically detach[49].This cell recovery tech-nique is a good alternative to the conventional,but often damaging,enzymatic or mechanical detachment methods. Recently,Hatakeyama et al.[41]have been producing bioactive,thermoresponsive cell culture surfaces by immobilizing the cell adhesive peptide RGDS and the growth factor insulin on a NiPAAm-copolymer.N-Iso-propylacrylamide was copolymerized with its analogue 2-carboxyisopropylacrylamide and the polymer was grafted onto polystyrene tissue culture dishes,followed by RGDS and insulin immobilization.They found that these factors increase cell adhesion and proliferation, reducing therefore culture time.When the temperature was brought to20°C,the cells could be easily recovered as contiguous tissue monolayers.
40L.Klouda,A.G.Mikos/European Journal of Pharmaceutics and Biopharmaceutics68(2008)34–45
4.PEO/PPO-based systems
Triblock copolymers poly(ethylene oxide)–b -poly(pro-pylene oxide)–b -poly(ethylene oxide)(PEO–PPO–PEO),known also as Pluronic òor Poloxamers,are another important group of synthetic polymers with a thermore-versible behavior in aqueous solutions.By adjusting the composition,the molecular weight and the concentration,this reversible gelation can occur at physiological tempera-ture and pH [50].The polymers owe their amphiphilic structure to the hydrophilic ethylene oxide and the hydro-phobic propylene oxide.The gelation mechanism of PEO/PPO block copolymers in aqueous solutions has been a topic of extensive investigation,and a possible explanation to this phenomenon could be given by the changes in micel-lar properties as a function of both concentration and temperature.Amphiphilic block copolymer molecules can self assemble into micelles in aqueous solutions.Above a certain concentration,termed as the critical micelle concen-tration (CMC),the polymer molecules,which were previ-
ously in solution,aggregate and form micelles.Members of the Pluronic òfamily which are used for drug delivery exhibit a CMC of 1l M to 1mM at 37°C.Moreover,the micelle formation has strong temperature dependence.Below a certain temperature,termed as the critical micelle temperature (CMT),both ethylene and propylene oxide blocks are hydrated and poly(propylene oxide)is relatively soluble in water.With temperature increase,poly(propyl-ene oxide)chains become less soluble,resulting in micelle formation [16].
As Pluronic òare commercially available in a range of molecular weights,composition ratios and forms,it would be useful to mention the nomenclature rules for these copolymers.The letter in the notation stands for liquid (L),paste (P)or ?akes (F),whereas the ?rst two numbers indicate the molecular weight of the PPO block and the last number the weight fraction of the PEO block.For exam-ple,the commonly used in biomedical applications F127has a weight percentage of 70%PEO and a molecular weight of PPO around 4000[50].
Over the past years,these copolymers have been exten-sively used in applications such as drug and gene delivery [16],inhibition of tissue adhesion [51,52]and burn wound covering [53,54].Newer advances in gene delivery are sum-marized elsewhere [55].Pluronic òrepresent a bio-inert environment,attributed by the hydrophilicity and ?exibil-ity of PEO chains [56].Therefore,most cells do not grow on these polymers,which have been used as tissue adhesion barriers.However,it was shown that Pluronic òcan be a good substrate for hematopoietic stem cells,supporting their culture and preservation more than conventional tis-sue culture dishes [57,58].Recently,the use of Pluronic òF127(synonymous to poloxamer 407)was reported for tis-sue engineering applications.This polymer has been found to have a rapid gelation at 37°C (after 1-min incubation,30%solution in cell culture medium)[59].F127was evalu-ated as a sca?old for lung tissue engineering,showing promising results on tissue growth with low in?ammatory response [60].Weinand and colleagues [59]tested a b -trical-cium phosphate (b -TCP)sca?old,using a F127hydrogel to facilitate cell delivery and distribution for an in vitro study aiming at bone regeneration.They reported that F127was no longer present in the channels of the b -TCP sca?old after 1week in culture and seemed to have degraded.Bone tissue growth was only weakly induced,and the constructs showed lower sti?ness than other hydrogel (?brin,collagen I)composites evaluated.
In general,Pluronic òF127has been found to have inad-equate mechanical integrity which makes them inappropri-ate for certain biomedical applications.The hydrogels show a low viscosity,which has as consequences poor mechanical strength,high permeability and limited stability with quick dissolution [61,62].Cohn and colleagues pro-posed two new mechanisms to create copolymers based on Pluronic òF127with improved mechanical properties.In both cases they relied on the principle of a multiblock backbone with the addition of covalently bound
repeating
Fig. 5.Drug release from a thermosensitive polymer micelle upon temperature increase.Reproduced from [37]with permission.
L.Klouda,A.G.Mikos /European Journal of Pharmaceutics and Biopharmaceutics 68(2008)34–4541
units.This way,the macromolecular structure and orienta-tion could be controlled.The?rst involved the polymeriza-tion of F127,with hexamethylene diisocyanate as a chain extender,forming poly(ether-urethanes).The second relied on the covalent binding of poly(ethylene glycol)and poly(propylene glycol),which as such do not possess ther-mogelling properties at physiological temperatures,using phosgene as a coupler and forming poly(ether-carbonates). Both newly synthesized polymers exhibited signi?cantly higher viscosities than F127at37°C,and the poly(ether-urethanes)displayed much slower drug release kinetics than the original polymer[61].The group developed the idea of thermoresponsive PEO/PPO polymers with improved mechanical behavior further in the next years. Their strategies included:(i)introduction of end groups that would allow for in situ chemical crosslinking after thermal gelation,such as carbon–carbon double bonds
[63],ethoxysilane groups[62,64]and methacrylate groups
[64],(ii)synthesis of poly(ethylene oxide)and poly(propyl-ene oxide)block copolymers using diacyl chloride as a cou-pling agent[65],(iii)synthesis of PEO/PPO copolymers with incorporation of e-caprolactone[65,66]or lactide [66]oligoester segments prior to chain extension.The latter approach yielded biodegradable hydrogels due to hydro-lytic cleavage of the ester bonds.
The need for more stable hydrogels was identi?ed also by Cellesi et al.[67–69].Their approach mimicked the nat-ural thermal gelation of alginate by relying on the occur-rence of a physical mechanism,attributed by the thermosensitive nature of Pluronicò,followed by an irre-versible chemical mechanism,due to covalent crosslinking by the reaction of groups at the termini of the copolymer. They named their gelation approach‘‘tandem process’’due to the cooperative action of both mechanisms(Fig.6).
Pluronicòpolymers were functionalized with acrylic moieties and thiols at their end groups and were subse-quently gelled at37°C,where a Michael-type addition took place and allowed for a slower chemical curing.It was found that these polymers were biocompatible,and so was their gelation process,which can be performed at physiological temperature and pH,allowing for encapsula-tion of sensitive drugs and cells[67].In order to limit steric hindrance phenomena,a similar method was followed with Tetronicòpolymers,which are thermosensitive tetra-armed Pluronicòanalogues[68].By adjusting the molecular weight of the precursors and the functionalization(there-fore also the crosslinking density),the?nal mechanical and transport properties of the‘‘tandem’’polymers can be controlled[68,69].Moreover,the‘‘tandem’’method allows for easy processing of the polymers,for example into spherical beads and hollow capsules[69].
5.Other synthetic polymers
5.1.PEG/biodegradable polyester copolymers
The copolymerization of hydrophilic,biocompatible poly(ethylene glycol)(PEG)with biodegradable and bio-compatible polyesters has yielded some interesting hydro-gel systems.Thermoresponsive properties were given by the appropriate adjustment of the hydrophobic polyester block and the PEG block length.
In1997,Jeong and colleagues[70]reported the synthesis of injectable poly(ethylene glycol)–b-poly(D,L-lactic acid-co-glycolic acid)–b-poly(ethylene glycol)(PEG–PLGA–PEG) triblock copolymers.These polymers were biocompatible, biodegradable and exhibited a sol–gel transition.The use of high molecular weight-PLGA combined with low molec-ular weight-PEG resulted in a hydrogel with quick gelation at physiological temperature.The combination of hydro-phobic/hydrophilic units created a surfactant behavior of the polymers in water,facilitating thus also the solubiliza-tion of hydrophobic drugs.In vivo studies showed su?cient mechanical properties and integrity for longer than a month[71].More recently,Chen et al.[72]developed a tri-block PLGA-PEG-PLGA-based system for the controlled release of testosterone.Testosterone is water-insoluble and so far,its delivery systems included patches,creams, gels,injectables and implants[73].A slower in vitro release of testosterone was observed for copolymers with longer PLGA blocks,possibly due to the slower degradation of these hydrophobic units.The thermosensitive polymers showed a controlled,linear release for a period of3
months.
Fig.6.Illustration of the‘‘tandem’’gelation process.Reproduced from[69]with permission. 42L.Klouda,A.G.Mikos/European Journal of Pharmaceutics and Biopharmaceutics68(2008)34–45
Another recent approach towards a thermoresponsive system involved the synthesis of a multiblock copolymer with a biodegradable polyester.Alternating multiblock poly(ethylene glycol)/poly(L -lactic acid)(PEG/PLLA)copolymers were produced.It was shown that sol-to-gel transition was depending on both the total molecular weight (MW)and the MW of each building block.In vitro and in vivo gelation studies determined that a copolymer with a total MW of 6700Da and 600/1300(MW of PEG/PLLA blocks,respectively)holds potential as an injectable carrier for biomedical applications in terms of transition temperature and modulus at 37°C (Fig.7)[74].Our group has proposed the combination of methoxy poly(ethylene glycol)(mPEG)with poly(propylene fuma-rate)(PPF)and the synthesis of a mPEG–PPF–mPEG tri-block copolymer [75].Copolymers exhibited an LCST depending on the molecular weight of mPEG,and it was shown that their LCST was strongly in?uenced by the pres-ence of salts.Moreover,the presence of the fumarate dou-ble bonds on PPF can allow for chemical crosslinking,enhancing thus the stability of the hydrogels.Recently,this hydrogel was evaluated for articular cartilage tissue engi-neering [76].Chondrocytes were encapsulated in the hydro-gel at 37°C and subsequently tested for their phenotypic characteristics.It was found that chondrocytes cultured in PEG/PPF hydrogels proliferated and produced signi?-cant levels of proteoglycans and collagen type II,which are both markers of the chondrocytic phenotype.When compared to cells cultured in agarose and alginate hydro-gels,two materials widely studied for chondrocyte delivery,proliferation levels in PEG/PPF were similar,however pro-teoglycan and collagen production was lower.Supplement of the bone morphogenic protein 7in PEG/PPF hydrogels
was also shown to increase chondrocyte proliferation,but not proteoglycan synthesis.5.2.Poly(organophosphazenes)
Current advances on poly(organophosphazenes)include their use as drug [77,78]and cell [79]delivery systems.Poly(organophosphazenes)grafted with mPEG and amino acid esters were reported as a new class of biodegradable and thermosensitive polymers in 1999[48].Sohn and col-leagues [80]developed a correlation for the LCST of these polymers as a function of their molecular structure,which comprises hydrophilic (PEG)and hydrophobic (amino acid esters)side groups.The polymers showed a sustained release pro?le for both hydrophobic [77]as well as hydro-philic [78]drugs for over 3and 2weeks,respectively.Also their use as cell carriers holds promise,as shown recently.Hepatocytes cultured in poly(organophosphazene)hydro-gels were able to maintain good viability and liver-speci?c activity for a period of 4weeks [79].6.Conclusions
Research in the area of thermoresponsive polymers for drug and gene delivery as well as for tissue adhesion pre-vention and wound covering has been well established in the past years.More recently,hydrogels exhibiting a ther-mosensitive sol–gel behavior have been reported as cell carriers for tissue regeneration.Typically,aqueous solu-tions of hydrogels used in biomedical applications are liquid at ambient temperature and gel at 37°C.Poly(N -isopropylacrylamide)and its copolymers with other syn-thetic or natural polymers is one of the most investigated thermoresponsive systems.By the appropriate copolymer-ization,intrinsic drawbacks of pNiPAAm like its non-bio-degradability and mechanical properties may be improved.Moreover,researchers have achieved to resolve the instability problems of another popular system,Plu-ronic ò.It is important to recognize the properties of the system(s)the hydrogel will be in contact with (such as hydrophilic or hydrophobic drugs,cells)and also the way and locus of administration in order to optimize the result.Each application has di?erent requirements,and it is possible to tailor the hydrogel properties to match a speci?c use.Acknowledgments
Research in the area of synthetic polymers for biomedi-cal applications has been funded by the National Institutes of Health (Grant Nos.:R01DE15164and R01AR48756).References
[1]N.A.Peppas,A.G.Mikos,Preparation methods and structure of hydrogels,in:N.A.Peppas (Ed.),Hydrogels in Medicine and Pharmacy,vol.1,CRC Press,Boca Raton,Florida,1986,pp.
1–25.
Fig.7.In?uence of total molecular weight of PEG/PLLA multiblock copolymers on the sol–gel transition temperature and storage modulus in aqueous solutions.The modulus was determined for a polymer concen-tration of 25wt %.Reproduced from [74]ith permission.
L.Klouda,A.G.Mikos /European Journal of Pharmaceutics and Biopharmaceutics 68(2008)34–4543
[2]N.A.Peppas,Hydrogels,in:Biomaterials Science,An Introduction to
Materials in Medicine,Academic Press,San Diego,California,1996, pp.60–64.
[3]A.S.Ho?man,Hydrogels for biomedical applications,Adv.Drug
Deliv.Rev.43(2002)3–12.
[4]A.K.Burkoth,K.S.Anseth,A review of photocrosslinked polyan-
hydrides:in situ forming degradable networks,Biomaterials21(2000) 2395–2404.
[5]J.A.Burdick, D.Frankel,W.S.Dernell,K.S.Anseth,An initial
investigation of photocurable three-dimensional lactic acid based sca?olds in a critical-sized cranial defect,Biomaterials24(2003) 1613–1620.
[6]D.A.Ossipov,J.Hilborn,Poly(vinyl alcohol)-based hydrogels formed
by‘‘click chemistry’’,Macromolecules39(2006)1709–1718.
[7]X.Z.Shu,S.Ahmad,Y.Liu,G.D.Prestwich,Synthesis and
evaluation of injectable,in situ crosslinkable synthetic extracellular matrices for tissue engineering,J.Biomed.Mater.Res.79(2006)902–912.
[8]C.K.Kuo,P.X.Ma,Ionically crosslinked alginate hydrogels as
sca?olds for tissue engineering:part1.Structure,gelation rate and mechanical properties,Biomaterials22(2001)511–521.
[9]N.A.Peppas,Physiologically responsive gels,https://www.doczj.com/doc/be17695989.html,pat.
Polym.6(1991)241–246.
[10]N.A.Peppas,P.Bures,W.Leobandung,H.Ichikawa,Hydrogels in
pharmaceutical formulations,Eur.J.Pharm.Biopharm.50(2000)27–46.
[11]L.Li,H.Shan,C.Y.Yue,https://www.doczj.com/doc/be17695989.html,m,K.C.Tam,X.Hu,Thermally
induced association and dissociation of methylcellulose in aqueous solutions,Langmuir18(2002)7291–7298.
[12]Y.Y.Liu,Y.H.Shao,J.Lu¨,Preparation,properties and controlled
release behaviors of pH-induced thermosensitive amphiphilic gels, Biomaterials27(2006)4016–4024.
[13]X.Yin,A.S.Ho?man,P.S.Stayton,Poly(N-isopropylacrylamide-co-
propylacrylic acid)copolymers that respond sharply to temperature and pH,Biomacromolecules7(2006)1381–1385.
[14]H.G.Schild,Poly(N-isopropylacrylamide):experiment,theory and
application,Prog.Polym.Sci.17(1992)163–249.
[15]N.T Southall,K.A.Dill,A.D.J.Haymet,A view of the hydrophobic
e?ect,J.Phys.Chem.B106(2002)521–533.
[16]A.V.Kabanov, E.V.Batrakova,V.Y.Alakhov,Pluronicòblock
copolymers as novel polymer therapeutics for drug and gene delivery, J.Control.Release82(2002)189–212.
[17]C.Joly-Duhamel,D.Hellio,M.Djabourov,All gelatin networks:1.
Biodiversity and physical chemistry,Langmuir18(2002)7208–7217.
[18]B.Jeong,S.W.Kim,Y.H.Bae,Thermosensitive sol–gel reversible
hydrogels,Adv.Drug Deliv.Rev.54(2002)37–51.
[19]E.Ruel-Gariepy,J.C.Leroux,In situ forming hydrogels-review of
temperature-sensitive systems,Eur.J.Pharm.Biopharm.58(2004) 409–426.
[20]M.Takahashi,M.Shimazaki,J.Yamamoto,Thermoreversible
gelation and phase separation in aqueous methyl cellulose solutions, J.Polym.Sci.B39(2001)91–100.
[21]W.Liu,B.Zhang,W.W.Lu,X.Li,D.Zhu,K.De Yao,Q.Wang,C.
Zhao,C.Wang,A rapid temperature-responsive sol–gel reversible poly(N-isopropylacrylamide)-g-methylcellulose copolymer hydrogel, Biomaterials25(2004)3005–3012.
[22]S.E.Stabenfeldt, A.J.Garcia,https://www.doczj.com/doc/be17695989.html,Placa,Thermoreversible
laminin-functionalized hydrogel for neural tissue engineering,J.
Biomed.Mater.Res.77A(2006)718–725.
[23]M.C.Tate,D.A.Shear,S.W.Ho?man,D.G.Stein,https://www.doczj.com/doc/be17695989.html,Placa,
Biocompatibility of methylcellulose-based constructs designed for intracerebral gelation following experimental traumatic brain injury, Biomaterials22(2001)1113–1123.
[24]N.Bhattarai,F.A.Matsen,M.Zhang,PEG-grafted chitosan as an
injectable thermoreversible hydrogel,Macromol.Biosci.5(2005) 107–111.
[25]N.Bhattarai,H.R.Ramay,J.Gunn,F.A.Matsen,M.Zhang,PEG-
grafted chitosan as an injectable thermosensitive hydrogel for sustained protein release,J.Control.Release103(2005)609–624.[26]J.H.Cho,S.H.Kim,K.D.Park,M.C.Jung,W.I.Yang,S.W.Han,
J.Y.Noh,J.W.Lee,Chondrogenic di?erentiation of human mesen-chymal stem cells using a thermosensitive poly(N-isopropylacryla-mide)and water-soluble chitosan copolymer,Biomaterials25(2004) 5743–5751.
[27]J.M.Dang,D.D.N.Sun,Y.Shin-Ya,A.N.Sieber,J.P.Kostuik,
K.W.Leong,Temperature-responsive hydroxylbutyl chitosan for the culture of mesenchymal stem cells and intervertebral stem cells, Biomaterials27(2006)406–418.
[28]A.Chenite,C.Chaput,D.Wang,https://www.doczj.com/doc/be17695989.html,bes,M.D.Buschmann,
C.D.Hoemann,J.C.Leroux,B.L.Atkinson,F.Binette,A.Selmani,
Novel injectable neutral solutions of chitosan form biodegradable gels in situ,Biomaterials21(2000)2155–2161.
[29]K.E.Crompton,J.D.Goud,R.V.Bellamkonda,T.R.Gengenbach,
D.I.Finkelstein,M.K.Horne,J.S.Forsythe,Polylysine-functional-
ised thermoresponsive chitosan hydrogel for neural tissue engineer-ing,Biomaterials(2006),doi:10.1016/j.biomaterials.2006.08.044. [30]X.Zhang, D.Wu, C.C.Chu,Synthesis and characterization of
partially biodegradable,temperature and pH sensitive Dex-MA/ PNIPAAm hydrogels,Biomaterials25(2004)4719–4730.
[31]X.Huang,T.L.Lowe,Biodegradable thermoresponsive hydrogels for
aqueous encapsulation and controlled release of hydrophilic model drugs,Biomacromolecules6(2005)2131–2139.
[32]M.Shirakawa,K.Yamotoya,K.Nishinari,Tailoring of xyloglucan
properties using an enzyme,Food Hydrocoll.12(1998)25–28. [33]D.R.Nisbet,K.E.Crompton,S.D.Hamilton,S.Shirakawa,R.J.
Prankerd,D.I.Finkelstein,M.K.Horne,J.S.Forsythe,Morphology and gelation of thermosensitive xyloglucan hydrogels,Biophys.
Chem.121(2006)14–20.
[34]H.Yang,W.J.Kao,Thermoresponsive gelatin/monomethoxy
poly(ethylene glycol)-poly(D,L-lactide)hydrogels:formulation,char-acterization and antibacterial drug delivery,Pharm.Res.23(2006) 205–214.
[35]S.Ohya,T.Matsuda,Poly(N-isopropylacrylamide)(PNIPAM)-
grafted gelatin as thermoresponsive three-dimensional arti?cial extracellular matrix:molecular and formulation parameters vs.cell proliferation potential,J.Biomater.Sci.Polym.Ed.16(2005) 809–827.
[36]E.S.Gil,D.J.Frankowski,R.J.Spontak,S.M.Hudson,Swelling
behavior and morphological evolution of mixed gelatin/silk?broin hydrogels,Biomacromolecules6(2005)3079–3087.
[37]M.Nakayama,T.Okano,T.Miyazaki,F.Kohori,K.Sakai,M.
Yokoyama,Molecular design of biodegradable polymeric micelles for temperature-responsive drug release,J.Control.Release(2006), doi:10.1016/j.conrel.2006.07.007.
[38]D.C.Coughlan,F.P.Quilty,O.I.Corrigan,E?ect of drug physicho-
chemical properties on swelling/deswelling kinetics and pulsatile drug release from thermoresponsive poly(N-isopropylacrylamide)hydro-gels,J.Control.Release98(2006)97–114.
[39]K.Na,J.H.Park,S.W.Kim,B.K.Sun,D.G.Woo,H.M.Chung,
K.H.Park,Delivery of dexamethasone,ascorbate,and growth factor(TGF b-3)in thermo-reversible hydrogel constructs embedded with rabbit chondrocytes,Biomaterials27(2006) 5951–5957.
[40]M.C.Hacker,B.B.Ma,J.D.Kretlow,A.G.Mikos,Novel macromers
for the fabrication of injectable,calcium-binding hydrogels,Trans-actions of the31st Annual Meeting of the Society for Biomaterials, Pittsburgh,USA,April26–29,2006.
[41]H.Hatakeyama,A.Kikuchi,M.Yamato,T.Okano,Bio-function-
alized thermoresponsive interfaces facilitating cell adhesion and proliferation,Biomaterials27(2006)5069–5078.
[42]Y.Pei,J.Chen,L.Yang,L.Shi,Q.Tao,B.Hui,J.Li,The e?ect of
pH on the LCST of poly(N-isopropylacrylamide)and poly (N-isopropylacrylamide-co-acrylic acid),J.Biomater.Sci.Polym.
Ed.15(2004)585–594.
[43]X.Z.Zhang,Y.Y.Yang,T.S.Chung,K.X.Ma,Preparation and
characterization of fast response macroporous poly(N-isopropyl-acrylamide)hydrogels,Langmuir17(2001)6094–6099.
44L.Klouda,A.G.Mikos/European Journal of Pharmaceutics and Biopharmaceutics68(2008)34–45
[44]H.Feil,Y.H.Bae,J.Feijen,S.W.Kim,E?ect of comonomer
hydrophilicity and ionization on the lower critical solution temper-ature of N-isopropylacrylamide copolymers,Macromolecules26 (1993)2496–2500.
[45]X.M.Liu,L.S.Wang,L.Wang,J.Huang,C.He,The e?ect of salt
and pH on the phase-transition behaviors of temperature-sensitive copolymers based on N-isopropylacrylamide,Biomaterials25(2004) 5659–5666.
[46]Y.Y.Liu,X.D.Fan,B.R.Wei,Q.F.Si,W.X.Chen,L.Sun,pH-
responsive amphiphilic hydrogel networks with IPN structure:a strategy for controlled drug release,Int.J.Pharm.308(2006) 205–209.
[47]F.J.Xu,E.T.Kang,K.G.Neoh,pH-and temperature-responsive
hydrogels from crosslinked triblock copolymers prepared via consec-utive atom transfer radical polymerization,Biomaterials27(2006) 2787–2797.
[48]S.C.Song,S.B.Lee,J.I.Jin,Y.S.Sohn,A new class of biodegradable
thermosensitive polymers.I.Synthesis and characterization of poly(organophosphazenes)with methoxy-poly(ethylene glycol)and amino acid esters as side groups,Macromolecules32(1999) 2188–2193.
[49]T.Okano,N.Tamada,H.Sakai,Y.Sakurai,A novel recovery system
for cultured cells using plasma-treated polystyrene dishes grafted with poly(N-isopropylacrylamide),J.Biomed.Mater.Res.27(1993) 1243–1251.
[50]P.Alexandridis,T.A.Hatton,Poly(ethylene oxide)-poly(propylene
oxide)-poly(ethylene oxide)block copolymer surfactants in aqueous solutions and interfaces:thermodynamics,structure,dynamics and modeling,Colloids Surf.A96(1995)1–46.
[51]A.Steinleitner,https://www.doczj.com/doc/be17695989.html,mbert,C.Kazensky,B.Cantor,Poloxamer407
as an intraperitoneal barrier material fort he prevention of postsur-gical adhesion formation and reformation in rodent models for reproductive surgery,Obstet.Gynecol.77(1991)48–52.
[52]F.Ahmed,P.Alexandridis,H.Shankaran,S.Neelamegham,T The
ability of poloxamers to inhibit platelet aggregation depends on their physicochemical properties,Thromb.Haemost.86(2001)1532–1539.
[53]I.R.Schmolka,Arti?cial skin.I.Preparation and properties of
pluronic F-127gels for treatment of burns,J.Biomed.Mater.Res.6 (1972)571–582.
[54]R.M.Nalbandian,R.L.Henry,K.W.Balko, D.V.Adams,N.R.
Neuman,Pluronic F-127gel preparation as an arti?cial skin in the treatment of third-degree burns in pigs,J.Biomed.Mater.Res.21 (1987)1135–1148.
[55]A.Kabanov,J.Zhu,V.Alakhov,Pluronic block copolymers for gene
delivery,Adv.Genet.53(2005)231–261.
[56]A.Higuchi,K.Sugiyama,B.O.Yoon,M.Sakurai,M.Hara,M.
Sumita,S.Sugawara,T.Shirai,Serum protein adsorption and platelet adhesion on pluronic-adsorbed polysulfone membranes, Biomaterials24(2003)3235–3245.
[57]A.Higuchi,N.Aoki,T.Yamamoto,Y.Gomei,S.Egashira,Y.
Matsuoka,T.Miyazaki,H.Fukushima,Y.M.Lee,S.Jyujyoji,S.H.
Natori,Bio-inert surface of Pluronic-immobilized?ask for preserva-tion of haematopoietic stem cells,Biomacromolecules7(2006)1083–1089.
[58]A.Higuchi,N.Aoki,T.Yamamoto,T.Miyazaki,H.Fukushima,
T.M.Tak,S.Jyujyoji,S.Egashira,Y.Matsuoka,S.H.Natori, Temperature-induced cell detachment on immobilized pluronic sur-face,J.Biomed.Mater.Res.A79(2006)380–392.
[59]C.Weinand,I.Pomerantseva,C.M.Neville,R.Gupta,E.Weinberg,
I.Madisch,F.Shapiro,H.Abukawa,M.J.Troulis,J.P.Vacanti,
Hydrogel-b-TCP sca?olds and stem cells for tissue engineering bone, Bone38(2006)555–563.
[60]J.Cortiella,J.E.Nichols,K.Kojima,L.J.Bonassar,P.Dargon,A.K.
Roy,M.P.Vacant,J.A.Niles,C.A.Vacanti,Tissue-engineered lung:
an in vivo and in vitro comparison of polyglycolic acid and Pluronic F-127hydrogels/somatic lung progenitor cell constructs to support tissue growth,Tissue Eng.12(2006)1213–1225.
[61]D.Cohn,A.Sosnik,A.Levy,Improved reverse thermo-responsive
polymeric systems,Biomaterials24(2003)3707–3714.
[62]A.Sosnik,D.Cohn,Ethoxysilane-capped PEO-PPO-PEO triblocks:
a new family of reverse thermo-responsive polymers,Biomaterials25
(2004)2851–2858.
[63]A.Sosnik,D.Cohn,J.San Roman,G.A.Abraham,Crosslinkable
PEO-PPO-PEO-based reverse thermo-responsive gels as potentially injectable materials,J.Biomater.Sci.Polym.Ed.14(2003)227–239.
[64]D.Cohn,A.Sosnik,S.Garty,Smart hydrogels for in situ generated
implants,Biomacromolecules6(2005)1168–1175.
[65]A.Sosnik,D.Cohn,Reverse thermo-responsive poly(ethylene oxide)
and poly(propylene oxide)multiblock copolymers,Biomaterials26 (2005)349–357.
[66]D.Cohn,https://www.doczj.com/doc/be17695989.html,ndo,A.Sosnik,S.Garty,A.Levi,PEO-PPO-PEO-
based poly(ether ester urethane)s as degradable reverse thermo-responsive multiblock copolymers,Biomaterials27(2006)1718–1727.
[67]F.Cellesi,N.Tirelli,J.A.Hubbell,Materials for cell encapsulation
via a new tandem approach combining reverse thermal gelation and covalent crosslinking,Macromol.Chem.Phys.203(2002) 1466–1472.
[68]F.Cellesi,N.Tirelli,J.A.Hubbell,Towards a fully-synthetic
substitute of alginate:development of a new process using thermal gelation and chemical cross-linking,Biomaterials25(2004) 5115–5124.
[69]F.Cellesi,N.Tirelli,A new process for cell microencapsulation and
other biomaterial applications:thermal gelation and chemical cross-linking in‘‘tandem’’,J.Mater.Sci.Mater.Med.16(2005)559–565.
[70]B.M.Jeong,D.S.Lee,Y.H.Bae,S.W.Kim,Biodegradable block
copolymers as injectable drug-delivery systems,Nature388(1997) 860–862.
[71]B.M.Jeong,D.S.Lee,Y.H.Bae,S.W.Kim,In situ gelation of PEG-
PLGA-PEG triblock copolymer aqueous solutions and degradation thereof,J.Biomed.Mater.Res.50(2000)171–177.
[72]S.Chen,J.Singh,Controlled delivery of testosterone from smart
polymer solution based systems:in vitro evaluation,Int.J.Pharm.
295(2005)183–190.
[73]M.Zitzmann,E.Nieschlag,Hormone substitution in male hypogo-
nadism,Mol.Cell.Endocrinol.161(2000)73–88.
[74]J.Lee,Y.H.Bae,Y.S.Sohn,B.M.Jeong,Thermogelling aqueous
solutions of alternating multiblock copolymers of poly(L-lactic acid) and poly(ethylene)glycol,Biomacromolecules7(2006)1729–1734.
[75]E.Behravesh, A.K.Shung,S.Jo, A.G.Mikos,Synthesis and
characterization of triblock copolymers of methoxy poly(ethylene glycol)and poly(propylene fumarate),Biomacromolecules3(2002) 153–158.
[76]J.P.Fisher,S.Jo, A.G.Mikos, A.H.Reddi,Thermoreversible
hydrogel sca?olds for articular cartilage engineering,J.Biomed.
Mater.Res.A71(2004)268–274.
[77]G.D.Kang,S.H.Cheon,S.C.Song,Controlled release of doxoru-
bicin from thermosensitive poly(organophosphazene)hydrogels,Int.
J.Pharm.319(2006)29–36.
[78]G.D.Kang,S.H.Cheon,G.Khang,S.C.Song,Thermosensitive
poly(organophosphazene)hydrogels for a controlled drug delivery, Eur.J.Pharm.Biopharm.63(2006)340–346.
[79]K.H.Park,S.C.Song,Morphology of spheroidal hepatocytes within
injectable,biodegradable and thermosensitive poly(organophospha-zene)hydrogel as cell delivery vehicle,J.Biosci.Bioeng.101(2006) 238–242.
[80]Y.S.Sohn,J.K.Kim,R.Song, B.Jeong,The relationship of
thermosensitive properties with structure of organophosphazenes, Polymer45(2004)3081–3084.
L.Klouda,A.G.Mikos/European Journal of Pharmaceutics and Biopharmaceutics68(2008)34–4545
JOIN IN 学生用书1 Word List Starter Unit 1.Good afternoon 下午好 2.Good evening 晚上好 3.Good morning 早上好 4.Good night 晚安 5.Stand 站立 Unit 1 6.count [kaunt] （依次）点数 7.javascript:;eight [eit] 八 8.eleven [i'levn] 十一 9.four [f?:] 四 10.five [faiv] 五 11.flag [fl?g] 旗 12.guess [ges] 猜 13.jump [d??mp] 跳 14.nine [nain] 九 15.number ['n?mb?] 数字 16.one [w?n] 一 17.seven ['sevn] 七 18.six [siks] 六 19.ten [ten] 十 20.three [θri:] 三 21.twelve [twelv] 十二 22.two [tu:] 二 23.your [ju?] 你的 24.zero ['zi?r?u] 零、你们的 Unit 2 25.black [bl?k] 黑色26.blue [blu:] 蓝色 27.car [kɑ:] 小汽车 28.colour ['k?l?] 颜色 29.door [d?:] 门 30.favourite [feiv?rit]javascript:; 特别喜爱的 31.green [gri:n] 绿色 32.jeep [d?i:p] 吉普车 33.orange ['?:rind?] 橙黄色 34.pin k [pi?k] 粉红色 35.please [pli:z] 请 36.purple ['p?:pl] 紫色 37.red [red] 红色 38.white [wait] 白色 39.yellow ['jel?u] 黄色 Unit 3 40.blackboard ['bl?kb?:d] 黑板 41.book [buk] 书 42.chair [t???] 椅子 43.desk [desk] 桌子 44.pen [pen] 钢笔 45.pencil ['pensl] 铅笔 46.pencil case [keis] 笔盒 47.ruler ['ru:l?] 尺、直尺 48.schoolbag [sku:l] 书包 49.tree [tri:] 树 50.window ['wind?u] 窗、窗口 Unit 4 51.brown [braun] 棕色 52.cat [k?t] 猫
让多媒体技术为小学英语教学插上翅膀 摘要：随着科学技术的发展，多媒体技术应用的领域越来越广，在小学英语课堂中的应用给课堂带来了新的面貌，它以生动形象的画面、鲜明多姿的色彩、直观清楚的演示在教育领域逐渐取代传统教学方式，与传统教学方式相比较，多媒体教学有着无法比拟的优势。 关键词：多媒体技术小学英语课堂 正文： 科技日新月异，伴随着科技的进步，多媒体技术也日趋成熟，逐渐地应用到了教育领域，多媒体技术的使用，使得教育领域发生了巨大的变化。多媒体技术贯穿了现代教育中的各个方面，各个学科，新教学手段（媒介）取代旧的就学手段这是教育发展的必然要求，多媒体运用到课堂，使得教育更具时代色彩，这也是事物发展的客观规律。 多媒体技术运用到小学英语教学中，对此我有切身感受，它颠覆了我们传统的教学方式，使得过去单一的教学模式因注入科技成果的运用而发生质的飞跃，如今的英语课堂用“高效优质”、“多姿多彩”、“有声有色”、“精彩纷呈”等这样的词语来形容并不为过。 一、利用多媒体充分调动学生学习积极性 多媒体技术就是利用电脑把文字、图形、影象、动画、声音及视频等媒体信息都数位化，并将其整合在一定的交互式界面上，使电脑具有交互展示不同媒体形态的能力，多媒体技术具有声形俱全与图文并茂的特点，能使一些枯燥抽象的概念、复杂的变化过程、现象各异的运动形式直观而形象地显现在学生面前。。传统的教学手段只有简单的板书和口述，新课标要求教师的角色转换，传统教学中教师是主导，而新课标要求学生的主体地位，教师应该是课堂的组织者。而多媒体技术在英语课堂中的应用就是组织者教师与主体学生联系的一个纽带。而这个纽带不像传统教学的填鸭式教育手段，而是多姿多彩，有声有色的多媒体教学，这样的教学方式，能引起了学生们极大的学习兴趣，而兴趣是最好的老师，有了兴趣他们就愿意用心学，花时间去学，甚至自觉地学，这样久儿久之，就能将
小学英语自我介绍简单七篇 小学的时候我们就开始学习英语了,用英语进行自我介绍是我们值得骄傲的哦,以下是小编为大家带来的小学英语自我介绍简单,欢迎大家参考。 小学英语自我介绍1 Goodafternoon, teachers! Today, I`m very happy to make a speech here. Now, let me introduce myself. My name is ZhuRuijie. My English name is Molly. I`m 12. I come from Class1 Grade 6 of TongPu No.2 Primary School. I`m an active girl. I like playing basketball. Because I think it`s very interesting. I`d like to eat potatoes. They`re tasty. My favourite colour is green. And I like math best. It`s fun. On weekend, I like reading books in my room. I have a happy family. My father is tall and strong.My mother is hard-working and tall, too. My brother is smart. And I`m a good student. My dream is
to be a teacher, because I want to help the poor children in the future. Thank you for listening! Please remember me! 小学英语自我介绍2 Hello,everyone,Iamagirl,mynameisAliceGreenIaminClasstwoGrad efive,Iamveryoutgoing,Icandomanyhousework,suchascooking,swe epingthefloor,andIlikesinginganddancing,IamgoodatpiayingSoc cer,IhopeIcanmakegoodfriendswithyou.Thankyou! 小学英语自我介绍3 Hi, boys and girls, good morning, it is my plearsure to take this change to introduce myself, my name is ---, i am 10 years old. About my family, there are 4 people in my family, father, mother, brother and me, my father and mother are all very kind, welcome you to visit my family if you have time! I am a very outgoing girl(boy), I like reading、playing games and listening to music, of course, I also like to make friends with all the
小学生英语自我介绍演讲稿五篇 小学生英语自我介绍演讲稿（1） Good afternoon, teachers! My name is YangXiaodan. I`m 11 now. I`m from Class2 Grade 5 of TongPu No.2 Primary School. My English teacher is Miss Sun. She`s quiet and kind. She`s short and young. My good friend is ZhangBingbing. She`s 12. She`s tall and pretty. We`re in the same class. We both like English very much. I like painting , listening to music, playing computer games and reading books. My favourite food is chicken. It`s tasty and yummy. I often do my homework and read books on Saturdays. This is me. Please remember YangXiaodan. Thank you very much! 小学生英语自我介绍演讲稿（2） Hi everybody, my name is XX ,what’s your name? I’m 11 years old, how old are you? My favourite sport is swimming, what’s your favourite sport? My favourite food is hamburgers,what food do you like?
Join in 小学五年级英语教案 介休市宋古二中上站小学庞汝君 Unit 6 Friends 单元目标： 1、单词：sport music kite cap car book dog cat rat frog spider butterfly 2、句型：Who is your best friend ? （重点） How old is he ? When is his birthday ? What’s his favourite food ? 3、段落：介绍自己的好朋友 My best friend’s name is Toby . He is ten years old . His favourite colour is red . His birthday is in May . He has got a dog . 4、语法：第三人称的人称代词和物主代词（难点） 他he 他的his 她she 她的her 一般现在时第三人称单数动词+s
5、故事：Emma , Jackie and Diana . Are you all right ? What’s the matter ? Don’t be silly . We can play together . The first class 一、教学目标： 两个句型1、Who is your best friend ? 2、How old is he ? 二、教学过程： 1、Talk about your best friend . （1）教师说句子Who is your best friend ? My best friend is Anna. 让学生先领会句子的意思，然后模仿， 小组练习对话并上台表演。 （2）第二个句子How old is he ? He is nine years old . 象上面一样练习，等到学生掌握后把两个问句连起来做 问答操练。
让多媒体走进小学英语课堂 多媒体技术整合于英语学科教学，主动适应课程的发展，并与其和谐自然融为一体。作为课程改革不可或缺的条件，多媒体技术在教学中的飞速发展，必将使教师更新教学观念，营造学习环境，完善教学模式，变革评价方法，促进科学与人文统一，让课堂更精彩。那么，多媒体技术在小学英语教学过程中到底有什么作用呢？笔者认为主要表现在以下几个方面： 一、运用多媒体，激发学习兴趣 托尔斯泰说：“成功的教学所需要的不是强制而是激发学生兴趣。”培养学生学习英语的兴趣无疑是小学英语教学的关键所在。学生只有对自己感兴趣的东西才会开动脑筋、积极思考，外国学者在研究可靠学习发生的条件时指出：学生只能学到那些他们有兴趣的东西，在缺乏适当的环境﹑背景和铺垫的时候，学习不能发生。因此，在教学中要重视培养和引导学生学习英语的兴趣爱好。现代信息技术具有形象性﹑直观性﹑色彩鲜艳﹑图像逼真等特点，能将抽象的教学内容变成具体的视听形象，激发情趣，调动学生多种感官同时参与，从而激发学生的学习兴趣，使学生从多角度轻松舒畅地接受知识，并使其记忆长久。 二、运用多媒体，突破教学难点 小学生的思维正处在由具体形象向抽象思维过渡的时期，而有些教学内容之所以成为学生学习的难点就是因为缺乏语境而显得不具体。如果仅凭教师的描述和讲解，往往是教师花了很大精力，教学效果却事倍功半。而运用多媒体演示就能够突破时空限制，化静为动，使抽象的概念具体化、形象化，不仅突破了教材的难点，还可以加深学生对教学内容的理解和掌握，从而提高教学效率，达到事半功倍的效果。 例如：在教学“in、on、behind、under、near”这几个表示空间关系的方位介词时，我利用多媒体设计了这样的动画：通过点按鼠标，一只小猫一会儿跳到书桌上，一会儿藏在桌子里，一会儿蹦到书桌下，一会儿躲到桌子后，一会儿又窜到桌子旁。随着小猫的位置移动，电脑会问：Where is the cat？这样连续演示几次，学生通过观察、思考，就能快速地回答：It’s on the desk\in the desk\under the desk……，动画刺激了学生的感官，诱发了思维，难点不仅易于突出，更易于突破。 三、运用多媒体，提高创新能力 创新能力是未来人才的重要素质，是一个民族兴旺发达的灵魂。因此发挥每个学生潜在的创造因素，培养学生的创新能力，是当前教育工作者研究的重要课题。要想培养创新能力，就要力求使学生处于动眼、动手、动口的主体激活状态。多媒体技术对文本、图形、声音、动画和视频等信息具有集成处理的能力，使教学手段趋于全方位、多层次，它能加速学生感知过程，促进认识深化、加深理解、
小学生英文自我介绍及故事(精选多篇) 第一篇：小学生英文自我介绍 good afternoon everyone! my name is max. my chinese name is dong li zhen. i am six years old. i am in the primary school of jinling high school hexi branch.my family have four people. one,myfather, his name is dongning, he is a worker; two,my mother, her name is liqing,she is a housewife; three,my pet yoyo ,he is a dog; there is one more. now let me see….oh,yes,that is me! i love my family. i have many toys,a car ,a helicopter, two balls, three jumpropes. i like jumpropes best of all,because i jumprope very good. i have a pencilcase ,it’s bule. i have an brown eraser. i have five pencils,they are many colors, and one ruler,it’s white. i like english,i like speak english thank you! 第二篇：小学生英文自我介绍 自我介绍 各位老师好，我叫邵子豪，今年15岁，现在是红星海学校初中二年级学生。 我是一个善良、自信、幽默、乐于助人的阳光男孩，平时我喜欢和同学一起打篮球，还喜欢和爸爸谈古论今，我自引以为豪的是我有一个善解人意的妈妈和博学多才的爸爸，我为我生活在这样一个充满爱和民主的家庭中感到幸福！ hello , teachers: mynameisshaoziha o. i’mfifteenyearsold. istudy in hongxinghaimiddeleschool..iamingradetwonow. iamakind,confident,humourousandhelpfulboy..ilikeplaying basketballwithmyfriends. andialsoliketotalkaboutthehsitoryandthecurrent newswithmyfather. i amveryproudofmyfatherandmymotherbecauseoftheirknowledge.
五年级下英语月考试卷-全能练考-Join in剑桥英语 姓名：日期：总分：100 一、根据意思，写出单词。（10′） Fanny:Jackie, ________[猜] my animal,my ________最喜欢的 animal.OK? Jackie:Ok!Mm…Has it got a nose? Fanny:Yes,it’s got a big long nose,…and two long ________[牙齿]. Jackie:Does it live in ________[美国]? Fanny:No,no.It lives ________[在] Africa and Asia. Jackie:Can it ________[飞]? Fanny:I’m sorry it can’t. Jackie:Is it big? Fanny:Yes,it’s very big and ________[重的]. Jackie:What ________[颜色] is it?Is it white or ________[黑色]? Fanny:It’s white. Jackie:Oh,I see.It’s ________[大象]. 二、找出不同类的单词。（10′） （）1 A.sofa B.table C.check （）2 A.noodle https://www.doczj.com/doc/be17695989.html,k C.way （）3 A.fish B.wolf C.lion （）4 A.bike B.car C.write （）5 A.tree B.farm C.grass （）6 A.big B.small C.other ( ）7 A.eat B.listen C.brown
Join In剑桥小学英语(改编版)入门阶段Unit 1Hello,hello第1单元嗨,嗨 and mime. 1 听,模仿 Stand up Say 'hello' Slap hands Sit down 站起来说"嗨" 拍手坐下来 Good. Let's do up Say 'hello' Slap hands Sit down 好. 我们来再做一遍.站起来说"嗨"拍手坐下来 the pictures. 2 再听一遍给图画编号. up "hello" hands down 1 站起来 2 说"嗨" 3 拍手 4 坐下来说 3. A ,what's yourname 3 一首歌嗨,你叫什么名字 Hello. , what's yourname Hello. Hello. 嗨. 嗨. 嗨, 你叫什么名字嗨,嗨. Hello, what's yourname I'm Toby. I'm Toby. Hello,hello,hello.嗨, 你叫什么名字我叫托比. 我叫托比 . 嗨,嗨,嗨. I'm Toby. I'm Toby. Hello,hello, let's go! 我是托比. 我是托比. 嗨,嗨, 我们一起! Hello. , what's yourname I'm 'm Toby. 嗨.嗨.嗨, 你叫什么名字我叫托比.我叫托比. Hello,hello,hello. I'm 'm Toby. Hello,hello,let's go! 嗨,嗨,嗨.我是托比. 我是托比. 嗨,嗨,我们一起! 4 Listen and stick 4 听和指 What's your name I'm Bob. 你叫什么名字我叫鲍勃. What's your name I'm Rita. What's your name I'm Nick. 你叫什么名字我叫丽塔. 你叫什么名字我叫尼克. What's your name I'm Lisa. 你叫什么名字我叫利萨. 5. A story-Pit'sskateboard. 5 一个故事-彼德的滑板. Pa:Hello,Pit. Pa:好,彼德. Pi:Hello,:What's this Pi:嗨,帕特.Pa:这是什么 Pi:My new :Look!Pi:Goodbye,Pat! Pi:这是我的新滑板.Pi:看!Pi:再见,帕特! Pa:Bye-bye,Pit!Pi:Help!Help!pi:Bye-bye,skateboard! Pa:再见,彼德!Pi:救命!救命!Pi:再见,滑板! Unit 16. Let's learnand act 第1单元6 我们来边学边表演.
一、多媒体的设计要符合学生认知规律多媒体的设计应依据学生学习的需求来分析，并采用解决问题的最佳方案，使教学效果达到最优化。 多媒体教学的手段能化被动为主动，化生硬为生动，化抽象为直观，化理论为实践。 根据小学生的认知规律，他们缺乏坚实的理论基础，但直观的操作演示可以弥补这项弱点，可以帮助他们更好地理解和掌握。 对此，学者们已渐有共识这种直观生动的教学形式极大地激发了学生的学习兴趣，也引发了学生丰富的想象力和创造力。 在教学设计过程中，一方面，要运用多媒体的功能优势，将课本中抽象原理形象化，从而激发学生学习的兴趣，提高学习效果；另一方面必须让教学过程符合学生的认知规律，要多为学生创设动手操作的机会。 例如，在教学?一课时，我利用多媒体设计了一次动物聚会，森林里有很多小动物，有些通过图片来呈现，有些通过动画来呈现。 我带领学生边欣赏多媒体演示边复习了小动物的单词，然后引导学生猜每种小动物都来了几个，通过猜测的方式，强调复数的用法。 这里我使用了链接的方式，让学生像在欣赏魔术表演一样，使其出于好奇而变得主动。 通过设计符合学生知识基础和认知能力的多媒体教学内容，不仅能够激发学生学习的积极性和主动性，而且能够促进学生对知识的自我建构和内化。 此外，考虑到学生的年龄特点，在每节课的教学过程中我还格外重视评价的作用，而多媒体技术在这方面也表现出独特的优势。
不论是学习单词、句型，还是与电脑进行互动交流，每个知识点 都有图案背景，并提供过程性评价，随之出现的评价语言和动画无不让学生兴奋异常。 这种符合学生认知规律和个性化特征的内容设计与反馈，不仅使学生们感受到获得成功的喜悦，更达到了快乐交互学习的目的。 二、多媒体的应用要适时、适度和适量信息技术整合于课程不是 简单地将信息技术应用于教学，而是高层次地融合与主动适应。 必须改变传统的单一辅助教学的观点，创造数字化的学习环境，创设主动学习情景，创设条件让学生最大限度地接触信息技术，让信息技术成为学生强大的认知工具，最终达到改善学习的目的。 为保证多媒体与英语教学有效的融合，充分发挥多媒体技术应用的效果，在利用多媒体进行教学设计过程中，要遵循适时、适度和适量的原则，即在恰当的时间选择合适的媒体，适量地呈现教学内容。 在设计多媒体的过程中，要把握教学时机，不同的教学内容和教学环节，决定了多媒体运用的最佳时机也不同，精心策划才能妙笔生花，不能为使用多媒体而使用，不要一味追求手段的先进性、豪华性，而造成课堂教学过程中的画蛇添足。 这就要求教师熟悉多媒体的功能和常用多媒体的种类、特点，以便在教学设计和应用过程中将多媒体与课程学习进行有效地融合，最终提高课堂教学效果。 例如，在学习?这一知识点时，传统的英语课常常是借助钟表或模型来解释某一时刻，讲解整点、几点几分、差几分几点。
公司登记（备案）申请书 注：请仔细阅读本申请书《填写说明》，按要求填写。 □基本信息 名称 名称预先核准文号/ 注册号/统一 社会信用代码 住所 省（市/自治区）市（地区/盟/自治州）县（自治县/旗/自治旗/市/区）乡（民族乡/镇/街道）村（路/社区）号 生产经营地 省（市/自治区）市（地区/盟/自治州）县（自治县/旗/自治旗/市/区）乡（民族乡/镇/街道）村（路/社区）号 联系电话邮政编码 □设立 法定代表人 姓名 职务□董事长□执行董事□经理注册资本万元公司类型 设立方式 （股份公司填写） □发起设立□募集设立经营范围 经营期限□年□长期申请执照副本数量个
□变更 变更项目原登记内容申请变更登记内容 □备案 分公司 □增设□注销名称 注册号/统一 社会信用代码登记机关登记日期 清算组 成员 负责人联系电话 其他□董事□监事□经理□章程□章程修正案□财务负责人□联络员 □申请人声明 本公司依照《公司法》、《公司登记管理条例》相关规定申请登记、备案，提交材料真实有效。通过联络员登录企业信用信息公示系统向登记机关报送、向社会公示的企业信息为本企业提供、发布的信息，信息真实、有效。 法定代表人签字：公司盖章（清算组负责人）签字：年月日
附表1 法定代表人信息 姓名固定电话 移动电话电子邮箱 身份证件类型身份证件号码 （身份证件复印件粘贴处） 法定代表人签字：年月日
附表2 董事、监事、经理信息 姓名职务身份证件类型身份证件号码_______________ （身份证件复印件粘贴处） 姓名职务身份证件类型身份证件号码_______________ （身份证件复印件粘贴处） 姓名职务身份证件类型身份证件号码_______________ （身份证件复印件粘贴处）
小学三年级英语（上册）重要知识点归纳 一、单词 Unit 1学习文具：pen (钢笔) pencil (铅笔) pencil-case ( 铅笔盒) ruler(尺子) eraser(橡皮) crayon (蜡笔) book (书) bag (书包) sharpener (卷笔刀) school (学校) Unit 2身体部位：head (头) face( 脸) nose (鼻子) mouth (嘴) eye (眼睛)leg (腿) ear (耳朵) arm (胳膊)finger (手指) leg (腿) foot (脚)body (身体) Unit 3颜色：red (红色的) yellow (黄色的)green (绿色的)blue (蓝色的) purple (紫色的) white (白色的) black (黑色的) orange (橙色的) pink (粉色的)brown (棕色的) Unit 4动物：cat (猫)dog (狗)monkey (猴子)panda (熊猫)rabbit( 兔子) duck (鸭子)pig (猪)bird (鸟) bear (熊)elephant (大象)mouse (老鼠) squirrel (松鼠) Unit 5食物：cake (蛋糕)bread (面包) hot dog (热狗) hamburger (汉堡包) chicken (鸡肉)French fries (炸薯条)coke (可乐)juice (果汁)milk (牛奶) water (水)tea (茶) coffee (咖啡) Unit 6数字：one (一) two (二) three (三)four (四) five (五)six( 六)seven (七) eight (八) nine( 九) ten( 十)doll (玩具娃娃) boat (小船)ball (球) kite (风筝) balloon (气球) car (小汽车)plane (飞机) 二、对话 1、向别人问好应该说――A: Hello! (你好!) B: Hi! (你好!) 2、问别人的名字应该说-――A：What's your name? 你的名字是什么? B：My name's Chen Jie. 我的名字是陈洁。 3、跟别人分手应该说――A: Bye.\ Good bye!(再见) B: See you.(再见) \ Goodbye.(再见) 4、A: I have a pencil\ bag\ruler 我有一只铅笔\书包\尺子。 B: Me too . 我也有。 5、早上相见应该说-――A: Good morning. 早上好!
有限合伙企业登记注册操作指南 风险控制部 20xx年x月xx日
目录 一、合伙企业的概念 (4) 二、有限合伙企业应具备的条件 (4) 三、有限合伙企业设立具备的条件 (4) 四、注册有限合伙企业程序 (5) 五、申请合伙企业登记注册应提交文件、证件 (6) （一）合伙企业设立登记应提交的文件、证件： (6) （二）合伙企业变更登记应提交的文件、证件： (7) （三）合伙企业注销登记应提交的文件、证件： (8) （四）合伙企业申请备案应提交的文件、证件： (9) （五）其他登记应提交的文件、证件： (9) 六、申请合伙企业分支机构登记注册应提交的文件、证件 (9) （一）合伙企业分支机构设立登记应提交的文件、证件 (10) （二）合伙企业分支机构变更登记应提交的文件、证件： (10) （三）合伙企业分支机构注销登记应提交的文件、证件： (11) （四）其他登记应提交的文件、证件： (12) 七、收费标准 (12) 八、办事流程图 (12) （一）有限合伙企业创办总体流程图（不含专业性前置审批） (12) （二）、工商局注册程序 (15)
（三）、工商局具体办理程序（引入网上预审核、电话预约方式） (16) 九、有限合伙企业与有限责任公司的区别 (16) （一）、设立依据 (16) （二）、出资人数 (16) （三）、出资方式 (17) （四）、注册资本 (17) （五）、组织机构 (18) （六）、出资流转 (18) （七）、对外投资 (19) （八）、税收缴纳 (20) （九）、利润分配 (20) （十）、债务承担 (21) 十、常见问题解答与指导 (21)
小学简单英语自我介绍范文（精选3篇） 小学简单英语自我介绍范文 当换了一个新环境后，我们难以避免地要作出自我介绍，自我介绍可以满足我们渴望得到尊重的心理。但是自我介绍有什么要求呢？下面是收集整理的小学简单英语自我介绍范文，仅供参考，希望能够帮助到大家。 小学简单英语自我介绍1 Good afternoon, teachers! I`m very happy to introduce myself here. I`m Scott. My Chinese name is SunZijing. I`m 13. I`m from Class1 Grade 6 of TongPu No.2 Primary School. I have many hobbies, such as playing ping-pong, playing badminton, listening to music, reading magazines and so on. My favourite food is chicken. It`s tasty and yummy. So I`m very strong. I`m a naughty boy. I always play tricks. And I just like staying in my room and reading books. But I have a dream, I want to be a car-designer. Because I`m a car fan.This is me. Please remember SunZijing! Thank you very much! 小学简单英语自我介绍2 Goodafternoon, teachers! Today, I`m very happy to make a speech here. Now, let me introduce myself. My name is ZhuRuijie.
JOIN IN英语三年级下册课本重点Start unit 1 Words morning afternoon evening night 2 Sentences Good morning !早上好 Good afternoon !下午好 Good evening!晚上好 Good night!晚安 3 Phrases clap your hands 拍拍手 jump up high 往高跳 shake your arms and your legs 晃动你的胳膊和腿 bend your knees 弯曲你的膝盖 touch your toes 摸摸你的脚指
stand nose to nose 鼻子对鼻子站着 Unit 1 Pets 1 Words cat猫dog狗bird 鸟mouse老鼠fish鱼rabbit 兔子frog青蛙hamster仓鼠 budgie鹦鹉tiger老虎monkey 猴子panda熊猫giraffe 长颈鹿elephant 大象bear 熊run跑sit坐fly飞swim游泳roar吼叫eat吃 2 Grammar ★名词的复数:一般在词尾直接加s,不规则变化要牢记: fish-----fish mouse------mice 3 Sentences 1.Have you got a pet ? 你有宠物吗?Yes ,I have. 是的,我有。/No, I haven’t. 不,我没有。 2.2. What have you got ? 你有什么宠物吗?I’ve got a dog . / A dog. 我有一只狗。 3.What colour is the cat ? 你的猫是什么颜色的?It’s black. 它是黑色的。 What iswizard’s pet? 巫师的宠物是什么? 4.What is it ? 它是什么? It’s a rabbit .它是只兔子。
→客户提供：场所证明租赁协议身份证委托书三张一寸相片 →需准备材料：办理税务登记证时需要会计师资格证与财务人员劳动合同 →提交名称预审通知书→公司法定代表人签署的《公司设立登记申请书》→全体股东签署的《指定代表或者公共委托代理人的证明》（申请人填写股东姓名）→全体股东签署的公司章程（需得到工商局办事人员的认可）→股东身份证复印件→验资报告（需到计师事务所办理：需要材料有名称预审通知书复印件公司章程股东身份证复印件银行开具验资账户进账单原件银行开具询证函租赁合同及场所证明法人身份证原件公司开设临时存款账户的复印件）→任职文件（法人任职文件及股东董事会决议）→住所证明（房屋租赁合同）→工商局（办证大厅）提交所有材料→公司营业执照办理结束 →需带材料→公司营业执照正副本原件及复印件→法人身份证原件→代理人身份证→公章→办理人开具银行收据交款元工本费→填写申请书→组织机构代码证办
理结束 →需带材料→工商营业执照正副本复印件原件→组织机构正副本原件及复印件→公章→公司法定代表人签署的《公司设立登记申请书》→公司章程→股东注册资金情况表→验资报告书复印件→场所证明（租赁合同）→法人身份证复印件原件→会计师资格证（劳动合同）→税务登记证办理结束 →需带材料→工商营业执照正副本复印件原件→组织机构正副本原件及复印件→税务登记证原件及复印件→公章→法人身份证原件及复印件→代理人身份证原件及复印件→法人私章→公司验资账户→注以上复印件需四份→办理时间个工作日→办理结束 →需带材料→工商营业执照正副本复印件原件→组织机构正副本原件及复印件→公章→公司法定代表人签署的《公司设立登记申请书》→公司章程→股东注册资金情况表→验资报告书复印件→场所证明（租赁合同）→法人身份证复印件原件→会计师资格证（劳动合同）→会计制度→银行办理的开户许可证复印件→税务登记证备案办理结束
小学生英语自我介绍演讲稿带翻译 篇一：小学英语自我介绍演讲稿 Good morning everyone! Today I am very happy to make a speech（演讲） here. Now, let me introduce myself. My name is Chen Qiuxiang. I am from Dingcheng center Primary School. I live in our school from Monday to Friday. At school, I study Chinese, Math, English, Music, Computer, and so on. I like all of them. I am a monitor, so I often help my teacher take care of my class. I think I am a good help. And I like Monday best because we have English, music, computer and on Tuesdays. There are four seasons in a year , but my favourite season is summer. Because I can wear my beautiful dress . When I am at home, I often help my mother do some housework. For example, wash clothes , clean the bedroom, do the dishes and so on . and my mother says I am a good help, too. In my spare time, I have many hobbies. I like reading books , going hiking, flying kites .But I like singing best, and my favourite English song is “The more we get
Starter Unit Good to see you again知识总结 一. 短语 1. dance with me 和我一起跳舞 2. sing with me 和我一起唱歌 3. clap your hands 拍拍你的手 4. jump up high 高高跳起 5.shake your arms and your legs晃晃你的胳膊和腿 6. bend your knees 弯曲你的膝盖 7. touch your toes 触摸你的脚趾8. stand nose to nose鼻子贴鼻子站 二. 句子 1. ---Good morning. 早上好。 ---Good morning, Mr Li. 早上好，李老师。 2. ---Good afternoon. 下午好。 ---Good afternoon, Mr Brown. 下午好，布朗先生。 3. ---Good evening，Lisa. 晚上好,丽莎。 ---Good evening, Bob. 晚上好，鲍勃。 4. ---Good night. 晚安。 ----Good night. 晚安。 5. ---What’s your name? 你叫什么名字？ ---I’m Bob./ My name is Bob. 我叫鲍勃。 6. ---Open the window, please. 请打开窗户。 ---Yes ,Miss. 好的，老师。 7. ---What colour is it? 它是什么颜色？ 它是蓝红白混合的。 ---It’s blue, red and white. 皮特的桌子上是什么？ 8. ---What’s on Pit’s table? ---A schoolbag, an eraser and two books. 一个书包，一个橡皮和两本书。 9. ---What time is it? 几点钟？ 两点钟。 ---It’s two. 10.---What’s this? 这是什么？ ---My guitar. 我的吉他。
JOIN IN英语三年级下册 Start unit 1 Words morning afternoon evening night 2 Sentences Good morning !早上好Good afternoon ！下午好Good evening！晚上好 Good night！晚安 3 Phrases clap your hands 拍拍手 jump up high 往高跳 shake your arms and your legs 晃动你的胳膊和腿 bend your knees 弯曲你的膝盖 touch your toes 摸摸你的脚指 stand nose to nose 鼻子对鼻子站着 Unit 1 Pets 1 Words cat猫dog狗bird 鸟mouse老鼠fish鱼rabbit 兔子frog青蛙hamster仓鼠 budgie鹦鹉tiger老虎monkey 猴子panda熊猫giraffe 长颈鹿elephant 大象bear 熊run跑sit坐fly飞swim游泳roar吼叫eat吃 2 Grammar
★名词的复数：一般在词尾直接加s,不规则变化要牢记: fish-----fish mouse------mice 3 Sentences 1.Have you got a pet ? 你有宠物吗？ Yes ,I have. 是的,我有。/No, I haven’t. 不，我没有。 2.What have you got ? 你有什么宠物吗？I’ve got a dog . / A dog. 我有一只狗。 3.What colour is the cat ? 你的猫是什么颜色的？It’s black. 它是黑色的。 What is wizard’s pet? 巫师的宠物是什么？ 4.What is it ? 它是什么? It’s a rabbit .它是只兔子。 5.How many budgies /mice are there? 这里有多少只鹦鹉/老鼠？ There are + 数字budgies/mice. 这里有------只鹦鹉/老鼠。 6.Fly like a budgie. 像鹦鹉一样飞。Run like a rabbit. 像兔子一样跑。 Swim like a fish. 像鱼一样游泳。Eat like a hamster. 像仓鼠一样吃东西。 Sit like a dog. 像狗一样坐。Roar like a tiger. 像老虎一样吼叫。 7.What are in the pictures. 图片里面是什么？Animals. 动物。 8. What animals? 什么动物？ 9.How many pandas (elephants /bears/ giraffes/ monkeys/ budgies) are there?有多少.？ How many + 可数名词的复数形式 Unit 2 The days of the week